Development and Validation of Stability Indicating RP-HPLC Method for Estimation of Ketorolac and Fluoromethalone in Ophthalmic Formulations

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1 Sharma et al: Development and Validation of Stability Indicating RP-HPLC Method for Estimation of Ketorolac 3815 International Journal of Pharmaceutical Sciences and Nanotechnology Volume 10 Issue 5 September October 2017 Research Paper MS ID: IJPSN MAITRI SHARMA Development and Validation of Stability Indicating RP-HPLC Method for Estimation of Ketorolac and Fluoromethalone in Ophthalmic Formulations Maitri V. Sharma 1, Nisha D. Patel 1 *, Bhavesh Prajapati 2 and Samir K. Shah 3 1 Department of Quality Assurance, Sardar Patel College of Pharmacy, Vadtal Road, Bakrol, Anand, Gujarat. India. 2 Department of Pharmaceutical Chemistry, Sardar Patel College of Pharmacy, Vadtal Road, Bakrol, Anand, Gujarat. India. 3 Principal, Department of Pharmacology, Sardar Patel College of Pharmacy, Vadtal Road, Bakrol, Anand, Gujarat, India. Received May 19, 2017; accepted July 13, 2017 ABSTRACT A simple, specific, accurate and stability-indicating reversed phase high performance liquid chromatographic method was developed for the simultaneous determination of ketorolac and fluorometholone in its ophthalmic formulation. ODS-BP hyperchrome C18 having 250 mm 4.6 mm 5μm with mobile phase composed of 0.2% Formic acid and 0.2% of TEA in water (ph adjusted to 5.0 with Formic Acid): Methanol (40:60 v/v) at flow rate of 1.0 ml/min, detection wavelength at 230 nm. The retention KEYWORDS: Ketorolac, Fluorometholone, Degradation, RP-HPLC; Opthalmic. times of ketorolac and fluorometholone were found to be min and min, respectively. Linearity was established for Ketorolac and fluorometholone in the range of μg/ml and μg/ml respectively. The percentage recoveries for ketorolac and fluorometholone were found to be in the range of 98-99% and 98-99% respectively. The LOD and LOQ for Ketorolac was found to be 5.14 and 15.5 μg/ml, and for fluorometholone 0.72 and 2.21 respectively. Introduction Ketorolac (KT), (±)-5-benzoyl-2,3-dihydro-1Hpyrrolizine-1-carboxylic acid, 2-amino-2- (hydroxymethyl)-1,3- propanediol is white or almost white, crystalline powder soluble in water and in methanol, slightly soluble in ethanol, practically insoluble in methylene chloride. Ketorolac is a non-steroidal antiinflammatory drug (NSAID) in the family of heterocyclic acetic derivatives, used as an analgesic. It is official in Indian Pharma-copoeia. Ketorolac in combine with fluorometholone is used in conjunctivitis (Heafindal et al, 2006; Brunton et al., 2010). The structure of Ketorolac is shown in Fig. 1. of archidonic acid from phospholipid. Therefore, it inhibits first step of synthesis of inflammatory mediator like prostaglandin, Bradykinin and Leukotriene. It is used as corticosteroid after laser-based refractive surgery. It is official in BP and USP. The structure of fluorometholone is shown in Fig. 2. Fig. 1. Chemical structure of ketorolac. Fluorometholone (FL), (1R, 2S, 8S, 10S, 11S, 14R, 15S, 17S)-14- acetyl-1-fluoro-14, 17-dihydroxy-2, 8, 15- trimethyltetracyclo [ , 7.011, 15] hepta deca-3, 6 dien-5-one is white to yellow white crystalline powder soluble in methanol, insoluble in water, slightly soluble in ethanol and ether. Fluorometholone inhibit synthesis Fig. 2. Chemical structure of fluorometholon. Ketorolac (KT) and fluorometholonene (FL) combination is available as eye drops for the treatment of conjunctivitis. The literature survey of ketorolac and fluorometholone reveals that various analytical methods are available for determination of ketorolac and fluorometholone individually and in other combinations (Vladimirov et al., 1996; Fan et al., 2007; Dubey et al., 2013; Bhavsar et al., 2013). Moreover, various analytical 3815

2 3816 Int J Pharm Sci Nanotech Vol 10; Issue 5 September October 2017 methods like RP-HPLC, and spectrophotometric methods like absorbance ratio method and simultaneous equation method are also reported in literature for determination of ketorolac and fluorometholone (Patel et al., 2013; Shah and Maheshwari, 2014). However, no stability indicating RP-HPLC method is reported in literature. So in present study it was decided to develop stability indicating RP- HPLC method for simultaneous estimation of Ketorolac and Fluorometholonene in ophthalmic formulation. The method was validated in compliance with ICH guidelines (ICH, 2006). Materials and Methods Materials: KETOROLAC and FLUOROMETHOLONE drugs were kindly gifted by Intas pharmaceuticals, Ltd. Baddi, Himachal Pradesh. All solvents and chemicals used were of analytical grade or HPLC grade purchased from Merck and Spectrochem. Marketed dosage forms used in this study was EYETRUST procured from local market. Equipment: The liquid chromatographic system was of Analytical Technologies which consisted of following components: A column C 18 (250 mm 4.6 mm, 5μm), S 1122 Pump, 2203 UV-Visible detector and loop injector. The chromatographic analysis was performed using Alchrome A 2000 software. Chromatographic conditions: The separation was achieved on C 18 (250 mm 4.6 mm, 5μm) column with mobile phase containing 0.2% Formic acid + 0.2% of TEA in Water (ph adjusted to 5.0 with Formic Acid): Methanol (40:60%v/v) at a flow rate of 1.0 ml/min. The eluted compounds were monitored at the wavelength of 230nm. The sample injection volume was 20 μl and total run time was 10 min. Preparation of Mobile Phase Accurately transferred 0.2% Formic acid and 0.2% Triethylamine (TEA) were prepared by taking 2 ml Formic acid and 2 ml (TEA) in 1000 ml volumetric flask and diluted upto 1000 ml with distilled water than take 400 ml solution and ph adjusted to 5.0 with Formic Acid and 600 ml of methanol (40:60, v/v) and used as a mobile phase. Selection of Wavelength for Determination Standard solution of FL (1μg/mL) and KT (5 μg/ml) were prepared by taking from their working standard solutions separately in each 10 ml volumetric flask. Make up volume using diluent as a solvent. Each solution was scanned between nm using water as a blank. The point at which both drug show absorbance was selected as wavelength for determination. Preparation of Standard Stock Solution of KT and FL Accurately weighed 100 mg of KT and 100 mg FL were transferred into 100 ml volumetric flask, dissolved and diluted up to mark with diluent to give a stock solution having strength of 1 mg/ml (1000μ/mL). Preparation of Working Standard Solution of KT Accurately transferred 10 ml KT solution taken from above stock solution and diluted with 100 ml the diluent having strength of 0.1mg/mL (100μ/mL). Preparation of Working Standard Solution of FL Accurately transferred 2 ml FL solution taken from above stock solution and diluted with 100 ml the diluent having strength of 0.02mg/mL (20μ/mL). Preparation of Combine Standard Stock Solution of KT and FL Accurately weighed FL (20 mg) and KT (100 mg) were transferred into 100 ml volumetric flask and dissolved in diluent to give a stock solution 200 μg/ml of FL and 1000 μg/ml of KT. Stock solution (1.0 ml) was transferred in 10 ml volumetric flask and diluted up to mark with diluent to obtain working standard solution 20 μg/ml of FL and 100 μg/ml of KT. This solution was used to prepare standard solution for linearity. Sample Preparation for Marketed Formulation (FL 0.1 mg/ml and KT 0.5 mg/ml) Based on above label claim sample prepared as follows: Accurately transferred 4 ml of marketed eye drop solution was into 20 ml volumetric flask and it was diluted up to mark with diluent to get final sample concentration of FL and KT to 20 μg/ml and 100 μg/ml respectively and injected. Forced Degradation Studies Preparation of Samples for Force Degradation Study Standard stock solution: Accuratey weighed quantity of KT 100 mg and FL 20 mg was transferred into 100 ml of volumetric flask, 70 ml of mobile phase added into it sonicated to dissolve, cooled and then dilute up to 100 ml of diluent. Further dilute 5.0 ml of this solution to 50 ml with Mobile Phase, mixed well. Sample preparation: Accurately transferred 4 ml of marketed drop solution into 20 ml volumetric flask and it was diluted up to mark with Mobile Phase to get final sample concentration of Fluorometholone and ketorolac to 20 μg/ml and 100 μg/ml respectively and injected. Acid Degradation Blank preparation for acidic degradation: Transferred 5 ml diluent in 20 ml volumetric flask and added 1 ml 1N HCl into it and it was kept at room temperature for 6 hours. Then added 1 ml of 1N NaOH to neutralize it and volume was made up to mark with Mobile Phase and mixed well. It was filtered through 0.45μm syringe filter. Sample preparation for acid degradation: 1 ml of standard stock solution (KT/FL/Sample) was transferred into 10 ml of volumetric flask. 1 ml of 1 N HCl solution was added and mixed well. The volumetric flask was kept in dark place for 6 hrs. After time period, mixture was neutralized by 1 ml of 1 N NaOH to stop reaction and then diluted to volume 10 ml with Mobile Phase.

3 Sharma et al: Development and Validation of Stability Indicating RP-HPLC Method for Estimation of Ketorolac 3817 Base Degradation Blank preparation for basic degradation: Transferred 5mL diluent in 20 ml volumetric flask and added 1 ml 1N NaOH into it and it was kept at room temperature for 6 hours. Then added 1 ml of 1N HCl to neutralize it and volume was made up to mark with mobile phase and mixed well. It was filtered through 0.45μ syringe filter. Sample preparation for base degradation: 1 ml of standard stock solution (KT/FL/Sample) was transferred in to 10 ml of volumetric flask. 1 ml of 1 N NaOH solution was added and mixed well. The volumetric flask was kept in dark place for 6 hrs. After time period, mixture was neutralized by 1 ml of 1 N HCl to stop reaction and then diluted to 10 ml with Mobile Phase. Oxidation Degradation Blank preparation for oxidation degradation: Transferred 5 ml mobile phase in 20 ml volumetric flask and added 5 ml 3% H 2 O 2 into it and it was kept at room temperature for 3 hours. Then volume was made up to mark with mobile phase and mixed well. It was filtered through 0.45μ syringe filter. Sample preparation for oxidation degradation: Accurately 4 ml of marketed drop solution was transferred into 20 ml volumetric flask, added 5 ml of 3% H 2 O 2 to it and it was kept for 3 hours at room temperature. Then volume was made upto mark with mobile phase and mixed well and injected. Photolytic degradation: Solution for photo degradation was prepared by dissolving 4 ml of marketed drop solution was transferred in 20 ml volumetric flask. This solution was kept in sunlight for 12 hours. Then volume was made up to mark with mobile phase and mixed well and 20 μl of this solution was injected in HPLC. Thermal degradation: Solution for thermal degradation was prepared by dissolving 4 ml of marketed drop solution was transferred into 20 ml volumetric flask. This solution was kept for 3 hours at 80 C temperature. Then volume was made up to mark with mobile phase and mixed well and 20 μl of this solution was injected. Method validation: As per ICH guidelines Q2R1, the method validation parameters studied were specificity, linearity, accuracy, precision, limit of detection and limit of quantification. Specificity: Specificity is ability to measure specifically the analyte of interest without any interferences from excipient and mobile phase component. For the determination of specificity 1 μg/ml solution of the standard FL and 5μg/mL solution of the standard KT was injected. Marketed formulation of same concentration was also injected. Both chromatograms were compared and checked for any interference of excipient peak. Chromatogram of blank (only mobile phase) was also recorded to check any interference. Single standard solutions of both drugs were injected for selectivity and peak information. Linearity (calibration curve) (n = 5): Mixed working standard solutions (4, 4.5, 5, 5.5 and 6 ml equivalent to 80, 90, 100, 110 and 120 μg/ml of KT and 16, 18, 20, 22 and 24 μg/ml FL) were transferred in a series of 10 ml volumetric flasks and diluted to the mark with diluent. The solutions of each concentration were injected under the operating chromatographic conditions as described earlier. Chromatograms were recorded. Calibration curves were constructed by plotting peak areas versus concentrations and the regression equations were calculated. These operations were done five times and mean responses were calculated. % RSD was calculated. It should not be more than 2%. Accuracy (% recovery): It was determined by calculating the recovery of KL and FL from formulation by standard addition method. To a fixed amount of 80%, 100% and 120% amount of standard was added and the amount of standard added was calculated using regression equation. Known amount of standard solutions of KT (80,100 and 120 μg/ml) and FL (16, 20 and 24 μg/ml) were added to a pre-quantified sample solution of KT and FL (100 and 20 μg/ml, respectively). Each solution was injected in triplicate and the percentage recovery was calculated by measuring the responses and fitting these values into the regression equations of the respective calibration curves. Precision Intraday precision: Sample of 4, 5 and 6 ml of working standard solution of KT (100 μg/ml) and FL (20 μg/ml) were transferred to a series of 10 ml volumetric flask. The volume was adjusted up to mark with water to get 80, 100 and 120 μg/ml solution of KT and 16, 20 and 24 μg/ml solution of FL. The area of peaks were measured three different times on the same day and % RSD was calculated. Interday precision: Aliquots of 4, 5 and 6 ml of working standard solution of KT (100 μg/ml) and FL (20 μg/ml) were transferred to a series of 10 ml volumetric flask. The volume was adjusted up to mark with water to get 80, 100 and 120 μg/ml solution of KT and 16, 20 and 24 μg/ml solution of FL. The area of peaks were measured three times on the three different days and % RSD was calculated. LOD and LOQ: The LOD (Limit of Detection) was estimated from the set of 5 calibration curves used to determine method linearity. The LOD may be calculated as LOD = 3.3 σ/s Where, σ = Standard deviation of the Y- intercepts of the 5 calibration curves S = Mean slope of the 5 calibration curves The LOQ (Limit of Quantitation) was estimated from the set of 5 calibration curves used to determine method linearity. The LOQ may be calculated as LOQ = 10 σ/s Where, σ = Standard deviation of the Y- intercepts of the 6 calibration curves S = Mean slope of the 6 calibration curves

4 3818 Int J Pharm Sci Nanotech Vol 10; Issue 5 September October 2017 Robustness: Robustness was performed by changing flow Rate, ph and mobile phase. 1. Change in Flow rate: 0.9 ml/min, 1.1mL/min 2. Change in mobile phase composition: Mobile Phase (Buffer: Solvent 40:60) Mobile Phase: 38:62, Mobile Phase 42:52 3. Change in ph (ph 5.0): Buffer ph 4.8, Buffer ph 5.2 System suitability: System suitability shall be checked for the conformance of suitability and reproducibility of system for analysis. System suitability was measured by number of theoretical plates, resolution, retention time and tailing factor. Results and Discussion Selection of wavelength: For selection of wavelength both drugs were scanned in the range of nm. The drugs showed reasonably good absorbance at 230nm. Hence 230 nm was selected as a wavelength for estimation in HPLC. and Fluorometholonene (FL) in presence of degradation products. An isocratic method was employed using 0.2% Formic acid and 0.2% of TEA in water (ph adjusted to 5.0 with Formic Acid): Methanol (40:60 v/v) as mobile phase, ODS-BP hyperchrome C18 (250 mm 4.6 mm 5 μm) Column with flow rate of 1.0 ml/min on HPLC equipped with UV- Visible detector. Tailing was observed in peak for Ketorolac and Fluorometholonene, hence to reduce the tailing attempt was made with 0.2% Formic acid +0.2% of TEA in water (ph adjusted to 5.0 with formic acid): Methanol (40:60 v/v) (Flow rate: 1.0 ml/min) as mobile phase. In optimized mobile phase Ketorolac and Fluoro-metholonene peaks were well resolved from their degradation products. The retention time of Ketorolac and Fluorometholonene were about 5.16 min and 2.96 min respectively. Optimized Chromatographic condition Validation of RP-HPLC Method Specificity: There was no interference of placebo at the retention time of Standard and Sample TABLE1 Chromatographic condition. Mobile phase Column Column Temperature Injection volume Flow rate Wavelength Diluent (0.2% Formic acid + 0.2% of TEA) in Water ph adjusted to 5.0 with Formic Acid: Methanol (40:60%v/v) C 18, 250 mm 4.6 mm, 5μ Room Temperature 20 μl 1.0 ml/min 230 nm Water: Methanol (40:60%v/v) Fig. 3. Overlay spectra of KT (5μg/mL) and FL (1 μg/ml). Method development and optimization: The main objective of this stability indicating chromatographic method was to separate and quantitate Ketorolac (KT) Linearity (Calibration Curve) (n=5): The linearity study was carried out for both drugs at different concentration levels. The linearity of KT and FL was in the range of μg/mL and 16-24μg/mL respectively. % RSD of all results were less than 2%. Fig. 4. Chromatogram using Mobile Phase - : 0.2% Formic acid and 0.2% of TEA (ph adjusted to 5.0 with formic acid) in Water: Methanol (40:60) (Flow rate: 1.0mL/min).

5 Sharma et al: Development and Validation of Stability Indicating RP-HPLC Method for Estimation of Ketorolac 3819 Fig. 5. Chromatogram of blank. Fig. 6. Chromatogram of Standard solution of FL (20 μg/ml) alone. Fig. 7. Chromatogram of Standard solution of KT (100 μg/ml) alone. Fig. 8. Chromatogram of Sample Solution FL (20 μg/ml) and KT(100μg/mL).

6 3820 Int J Pharm Sci Nanotech Vol 10; Issue 5 September October 2017 Fig. 9. Chromatogram of Standard Solution FL(20 μg/ml) and KT(100μg/mL). Fig. 10. Overlay chromatogram of Mixture containing standard KT ( μg/ml) and standard FL (16-24 μg/ml). Fig. 11. Calibration curve of ketorolac. Fig. 12. Calibration curve of fluoromethalone.

7 Sharma et al: Development and Validation of Stability Indicating RP-HPLC Method for Estimation of Ketorolac 3821 Accuracy Accuracy of the method was confirmed by recovery study from marketed formulation at three level of standard addition. Precision All results were found within the %RSD limit. Variation in interday precision was higher than intraday precision. LOD and LOQ The LOD and LOQ were estimated from the set of five calibration curves. TABLE 2 Linearity data for KT and FL. Conc. (μg/ml) Mean area ± SD % RSD KT FL KT FL KT FL ± ± ± ± ± ± ± ± ± ± TABLE 3 Accuracy for KT (N=3). Amount of KT (μg/ml) 80 TABLE 4 Accuracy for FL (N=3). Amount of KT (μg/ml) 16 % of Std KT added % of Std KT added Total amount added Mean amount recovered (μg/ml) Robustness Robustness was performed by changing Flow Rate, ph and Mobile phase. System suitability Parameter The acceptance criteria were less than 2% relative standard deviation (RSD) for the peak area and retention time, column plates greater than 2000, tailing factor less than 1.5 and capacity factor greater than 3.0. The results of system suitability analysis as shown in table. Forced Degradation Study Significant degradation of KT and FL was observed in acid, base, oxidative, photolytic and thermla condition. Degradant peaks were well resolved in both the drugs. % Recovery (mean ± SD) % RSD 50% ± % ± % ± Total amount added Mean amount recovered (μg/ml) % Recovery (mean ± SD) % RSD 50% ± % ± % ± TABLE 5 Intraday Precision for FL and KT. Intraday Precision data (n=3) KT FL Conc. (μg/ml) Mean area ± SD % RSD Conc. (μg/ml) Mean area ± SD % RSD ± ± ± ± ± ± TABLE 6 Interday Precision for KT and FL. Interday Precision data (n=3) KT FL Conc. (μg/ml) Mean area ± SD % RSD Conc. (μg/ml) Mean area ± SD % RSD ± ± ± ± ± ± TABLE 7 LOD and LOQ for KT and FL. Parameter KT FL S.D. of the Y- intercepts of the 5 calibration curves Mean slope of the 5 calibration curves LOD = 3.3 (SD/Slope) (μg/ml) LOQ = 10 (SD/Slope) (μg/ml)

8 3822 Int J Pharm Sci Nanotech Vol 10; Issue 5 September October 2017 TABLE 8 Robustness data for KT and FL. KT FL Parameters Variation Mean ± SD %RSD Mean ± SD %RSD As Such ± ± Flow rate (1.0 ml/min) Mobile phase ph (5.0) Mobile phase Ratio (40:60) TABLE 9 System suitability for KT and FL. 0.9 ml/min ± ± ml/min ± ± ph (4.8) ± ± ph (5.2) ± ± : ± ± : ± ± S. No. Name Retention Time Area Tailling Factor Theoretical Plates Resolution 1 FL KT TABLE 10 Analysis of marketed formulation. Drugs Label claim (mg/ml) % Amount of drug found % RSD Fluorometholone % 0.5 Ketorolac % 0.3 TABLE 11 Summary of Validation Parameter. Parameters Ketorolac Fluorometholone Linearity (μg/ml) Precision (% RSD) Intraday (n=3) Interday (n=3) Accuracy (% Recovery) Robustness (% RSD) Flow rate ph Mobile Phase LOD (μg/ml) LOQ (μg/ml) Acid degradation Fig. 13. Blank Chromatogram of Acidic degradation. Fig. 14. Sample Chromatogram of FL.

9 Sharma et al: Development and Validation of Stability Indicating RP-HPLC Method for Estimation of Ketorolac 3823 Fig. 15. Sample Chromatogram of KT. Fig. 16. Chromatogram of standard KT and FL after Acid treatment. Base degradation Fig. 17. Blank Chromatogram of Basic Degradations. Fig. 18. Sample Chromatogram of FL. Fig. 19. Sample Chromatogram for KT after base treatment Fig. 20. Chromatogram of Standard KT and FL

10 3824 Int J Pharm Sci Nanotech Vol 10; Issue 5 September October 2017 Oxidative degradation Fig. 21. Blank Chromatogram for Oxidative Degradation Fig. 22. Sample Chromatogram for FL. Fig. 23. Sample Chromatogram for KT Fig. 24. Chromatogram of Standard KT and FL after Oxidative treatment Sunlight degradation Fig. 25. Sample Chromatogram for FL Fig. 26. Sample Chromatogram for KT.

11 Sharma et al: Development and Validation of Stability Indicating RP-HPLC Method for Estimation of Ketorolac 3825 Fig. 27. Chromatogram of Standard KT and FL after Sunlight treatment. Thermal Degradation Fig. 28. Sample Chromatogram for FL Fig. 29. Sample Chromatogram for KT. Fig. 30. Chromatogram of Standard KT and FL after Thermal treatment.

12 3826 Int J Pharm Sci Nanotech Vol 10; Issue 5 September October 2017 TABLE 12 Summary of forced degradation study Stress type Stress conditions Fluorometholone Ketorolac % Assay % Degradation % Assay % Degradation Control sample As such sample 99.4% NA 98.8% NA Acid degradation 1N HCl, 1mL at RT for 6 hours 87.0% 12.4% 85.4% 13.4% Base degradation 1N NaOH, 1mL at RT for 6 hours 86.3% 13.1% 82.7% 16.1% Peroxide degradation 5 ml 3% H 2 O 2 at RT for 3 hours 89.2% 10.2% 87.3% 11.5% Thermal degradation At 80 C for 3 hours 89.8% 9.6% 88.2% 10.6% Sunlight degradation sunlight for 12 hours 89.5% 9.9% 88.1% 10.7% Conclusions A simple stability indicating Reverse Phase High Performance Liquid Chromatographic method have been developed and validated as per ICH guideline. All parameters were found to be within the prescribed limits. Force degradation study was performed and it was concluded that KT was more degraded which compared to FL in all degradation condition. All degradation peaks were well separate from each other and main peak. Degradation peaks were not interfered with the main peak of standards. This stability indicating assay method can be used for the simultaneous estimation of ketorolac (KT) and Fluorometholone (FL) in pharmaceutical dosage Form. Acknowledgements The authors are thankful to Intas Pharmaceuticals Ltd, Baddi, Himachal Pradesh, India for providing Ketorolac and Fluorometholone as gift sample for this work. They also thank Sardar Patel College of Pharmacy for providing required facilities to carry out this research work. References Bhavsar DN, Thakor NM, Patel C, Shah VH and Upadhyay UM (2013). Simultaneous Estimation of Ketorolac Tromethamine and Omeprazole by Ultraviolet Spectroscopy. IJRPS 3: Brunton LL, Lazo JS and Parkt KL (2010). The pharmacological basis of therapeutics; 11 th ed. Goodman and Gilmans, Mc Graw Hill medical publishing, pp Dubey SK, Duddelly S, Jangala H and Saha RN (2013). Rapid and Sensitive Reverse-Phase High-Performance Liquid Chromatography Method for Estimation of Ketorolac in Pharmaceuticals Using Weighted Regression. Indian J Pharm Sci 75: Fan XU, Fang YU, Bei-chengshang and Gui-li XU (2007). Determination of Ketorolac in Human Plasma by HPLC. Chinese Journal of Hospital Pharmacy 27: 1-8. Heafindal ET, Helms RA, Gourley DR and Quan DJ (2006). Textbook of Therapeutics; 8th ed. Lippincott Williams and Wilkins, pp 278. ICH guideline Q2 (R1) (2006). Text on Validation of Analytical Procedure, Methodology International Conference on Harmonization. Geneva. Patel R, Chauhan P and Shah SK (2013). Quntification of Ketorolac and Fluorometholone by RP-HPLC method on ophthalmic formulation. International Journal of Pharmaceutical Research 6: Shah J and Maheshwari DG (2014). Development and Validation of first order derivative UV Spectrophotometric method for simultaneous estimation of Fluorometholone acetate and ketorolac in ophthalmic dosage form. IJPR 6: Vladimirov S, Cudina O, Agbaba D and Zivanov-stakic D (1996). Spectrophotometric Determination of Fluorometholone in Pharmaceuticals Using 1,4-dihydrazinophthalazine. Analytical Letters 29: Address correspondence to: Nisha D. Patel, Department of Quality Assurance, Sardar Patel College of Pharmacy, Vadtal Road, Bakrol, Anand , Gujarat, India. Mob: ; patelnisha8290@gmail.com